GENE THERAPY FOR GALACTOSEMIA

1 . A nucleic acid molecule, comprising an adeno-associated virus (AAV) expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′:

a 5′ AAV inverted terminal repeat (ITR);

a promoter;

a transgene encoding Galactose-1-Phosphate Uridylyltransferase (GALT); and

a 3′ AAV ITR,

wherein the transgene comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 1.

2 . The nucleic acid molecule of claim 1 , wherein the transgene comprises the nucleic acid sequence of SEQ ID NO: 1.

3 . The nucleic acid molecule of claim 1 or claim 2 , wherein the promoter is operably linked to the transgene.

4 . The nucleic acid molecule of claim 3 , wherein the promoter comprises a CB6 promoter.

5 . The nucleic acid molecule of claim 4 , wherein the CB6 promoter comprises the nucleic acid sequence of SEQ ID NO: 2, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 2.

6 . The nucleic acid molecule of any one of claims 1-5 , wherein the AAV expression cassette comprises a rabbit globin polyadenylation sequence.

7 . The nucleic acid molecule of claim 6 , wherein the rabbit globin polyadenylation sequence comprises the nucleic acid sequence of SEQ ID NO: 3, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 3.

8 . The nucleic acid molecule of any one of claims 1-7 , wherein the AAV expression cassette comprises a CMV-IE enhancer.

9 . The nucleic acid molecule of claim 8 , wherein the CMV-IE enhancer comprises the nucleic acid sequence of SEQ ID NO: 4, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 4.

10 . The nucleic acid molecule of any one of claims 1-9 , wherein the AAV expression cassette comprises a Kozak sequence.

11 . The nucleic acid molecule of claim 10 , wherein the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 5.

12 . The nucleic acid molecule of any one of claims 1-11 , wherein the 5′ AAV ITR sequence comprises the nucleic acid sequence of SEQ ID NO: 6, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 6.

13 . The nucleic acid molecule of any one of claims 1-12 , wherein the 3′ AAV ITR sequence comprises the nucleic acid sequence of SEQ ID NO: 7, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 7.

14 . The nucleic acid molecule of any one of claims 1-13 , wherein the AAV expression cassette comprises, from 5′ to 3′:

the 5′ AAV ITR,

a CMV-IE enhancer,

a CB6 promoter,

a Kozak sequence,

the transgene encoding GALT,

a rabbit globin polyadenylation sequence, and

the 3′ AAV ITR.

15 . The nucleic acid molecule of any one of claims 1-14 , wherein the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 8, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 8.

16 . A plasmid, comprising the nucleic acid molecule of any one of claims 1-15 .

17 . A cell, comprising the nucleic acid molecule of any one of claims 1-15 , or the plasmid of claim 16 .

18 . A method of producing a recombinant adeno-associated virus (rAAV), the method comprising:

contacting an AAV producer cell with the nucleic acid molecule of any one of claims 1-15 , or the plasmid of claim 16 .

19 . A recombinant adeno-associated virus (rAAV) produced by the method of claim 18 .

20 . The rAAV of claim 19 , wherein the rAAV comprises an AAV9 capsid protein, an AAV8 capsid protein and/or an AAVrh10 capsid protein.

21 . The rAAV of claim 20 , wherein the rAAV comprises an AAV9 capsid protein.

22 . A recombinant adeno-associated virus (rAAV), comprising:

an AAV9 capsid protein; and the nucleic acid molecule of any one of claims 1-15 .

23 . A recombinant adeno-associated virus (rAAV), comprising: an AAV9 capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′:

a 5′ AAV ITR comprising the nucleic acid sequence of SEQ ID NO: 6;

a CMV-IE enhancer comprising the nucleic acid sequence of SEQ ID NO: 4;

a CB6 promoter comprising the nucleic acid sequence of SEQ ID NO: 2;

a Kozak sequence comprising the nucleic acid sequence of SEQ ID NO: 5;

a transgene encoding GALT comprising the nucleic acid sequence of SEQ ID NO: 1;

a rabbit globin polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 3; and

a 3′ AAV ITR comprising the nucleic acid sequence of SEQ ID NO: 7.

24 . A recombinant adeno-associated virus (rAAV), comprising: an AAV9 capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′:

a 5′ AAV ITR comprising the nucleic acid sequence of SEQ ID NO: 6, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 6;

a CMV-IE enhancer comprising the nucleic acid sequence of SEQ ID NO: 4, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 4;

a CB6 promoter comprising the nucleic acid sequence of SEQ ID NO: 2, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 2;

a Kozak sequence comprising the nucleic acid sequence of SEQ ID NO: 5, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 5;

a transgene encoding GALT comprising the nucleic acid sequence of SEQ ID NO: 1, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 1;

a rabbit globin polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 3, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 3; and

a 3′ AAV ITR comprising the nucleic acid sequence of SEQ ID NO: 7, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 7.

25 . A recombinant adeno-associated virus (rAAV), comprising:

an AAV9 capsid protein; and

a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 8.

26 . A recombinant adeno-associated virus (rAAV), comprising:

an AAV9 capsid protein; and

a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 8, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater identity to SEQ ID NO: 8.

27 . The rAAV of any one of claims 19-26 , wherein the AAV expression cassette comprises an miR-1 binding site, an miR-133a binding site, and/or an miR-122 binding site.

28 . The rAAV of any one of claims 19-27 , wherein the rAAV is a self-complementary AAV.

29 . The rAAV of any one of claims 19-27 , wherein the rAAV is a single-stranded AAV.

30 . A pharmaceutical composition, comprising:

(a) the nucleic acid molecule of any one of claims 1-15 , the plasmid of claim 16 , the cell of claim 17 , or the rAAV of any one of claims 19 - 29 ; and

(b) a pharmaceutically acceptable carrier.

31 . A method of expressing Galactose-1-Phosphate Uridylyltransferase (GALT) in a cell, comprising:

contacting the cell with the nucleic acid molecule of any one of claims 1-15 , the plasmid of claim 16 , the rAAV of any one of claims 19 - 29 , or the composition of claim 30 , thereby expressing GALT in the cell.

32 . A method of reducing the level of galactose-1-phosphate in a cell, comprising:

contacting the cell with the nucleic acid molecule of any one of claims 1-15 , the plasmid of claim 16 , the rAAV of any one of claims 19 - 29 , or the composition of claim 30 , thereby reducing the level of galactose-1-phosphate in the cell.

33 . The method of claim 31 or 32 , wherein the contacting step is performed in vitro, ex vivo, or in vivo.

34 . The method of claim 33 , wherein the contacting step is performed in vivo in a subject in need thereof.

35 . The method of claim 34 , wherein the contacting step comprises administering a therapeutically effective amount of the nucleic acid molecule, the plasmid, the rAAV, or the composition to the subject.

36 . A method of treating galactosemia in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of the nucleic acid molecule of any one of claims 1-15 , the plasmid of claim 16 , the cell of claim 17 , the rAAV of any one of claims 19-29 , or the composition of claim 30 , thereby treating galactosemia in the subject.

37 . The method of claim 35 or 36 , wherein the nucleic acid molecule, the plasmid, the cell, the rAAV, or the composition is administered intravenously.

38 . The method of any one of claims 35-37 , wherein the method comprises administering a therapeutically effective amount of rAAV, wherein the therapeutically effective amount is in a range of 10 10 genome copies to 10 14 genome copies per kilogram.

39 . The method of any one of claims 35-38 , wherein the administration results in an increase in the level of GALT in a cell of the subject, as compared to a control subject having galactosemia who is not administered the nucleic acid molecule, the plasmid, the cell, the rAAV or the composition.

40 . The method of any one of claims 35-39 , wherein the administration results in a decrease in the level of galactose-1-phosphate in a cell of the subject, as compared to a control subject having galactosemia who is not administered the nucleic acid molecule, the plasmid, the cell, the rAAV or the composition.

41 . The method of claim 39 or 40 , wherein the level of GALT is increased and/or the level of galactose-1-phosphate is decreased in a brain cell, a liver cell, an ovarian cell, or a red blood cell (RBC).

42 . The method of any one of claims 35-41 , wherein the administration diminishes the severity of a symptom of galactosemia.

43 . The method of any one of claims 35-42 , wherein the administration delays the onset of a symptom of galactosemia.

44 . The method of any one of claims 35-43 , wherein the administration eliminates a symptom of galactosemia.

45 . The method of any one of claims 42-44 , wherein the symptom of galactosemia is liver dysfunction, kidney dysfunction, sepsis, brain edema, pseudotumor cerebri, feeding difficulty, reduction in growth, cataracts, speech disorder, language disorder, dyspraxia, developmental delay, attention deficit, learning delay, neurological impairment, behavioral disorder, emotional disorder, tremors, fertility dysfunction, primary ovarian insufficiency, anxiety, seizures, depression, attention deficit hyperactivity disorder (ADHD), dementia, or any combination thereof.

46 . The method of any one of claims 36-45 , wherein the galactosemia is classic type I galactosemia.

47 . The method of any one of claims 34-46 , wherein the subject is a human subject.

48 . The method of claim 47 , wherein the subject is a neonate or an infant.

49 . The method of any one of claims 34-48 , wherein the subject suffers from galactosemia.

50 . The method of any one of claims 34-49 , wherein the subject has been diagnosed with galactosemia.

51 . The method of any one of claims 34-49 , wherein the subject has one or more mutations in the GALT gene.

52 . The method of any one of claims 34-51 , wherein the subject is at a risk of developing at least one symptom of galactosemia.